Technical Field
The present invention generally relates to methods for processing samples for molecular diagnostic applications.
Description of the Related Art
Many diagnostic, research, and development procedures require the detection of specific nucleic acid (DNA or RNA) sequences present in a biological sample. For example, nucleic acid detection methods are used to identify bacteria, viruses, or other microorganisms, whose presence can indicate the cause of infectious disease. Nucleic acid biomarkers are the target analytes for several infectious diseases of high global health importance, including human immunodeficiency virus (HIV), Hepatitis C virus (HCV), Hepatitis B virus (HBV), pandemic influenza, and dengue. The nucleic acids of more complex specimens, such as human tissues, are also more and more commonly tested in order to establish the presence of a mutation associated with cancer or a genetic disease. Detection of nucleic acids in samples of biological tissue, such as blood taken directly from a crime scene, is also used to determine the identity of the individual from which the sample originated, as, for example, paternity testing or forensic analysis.
Before a nucleic acid molecule can be detected for any of the purposes noted above, it is necessary to make the specific nucleic acid of interest available from a sample of biological material. The process of releasing nucleic acids from a biological specimen is commonly known in the art as “sample preparation”. Frequently, the target nucleic acid will be contained within a viral particle, a bacterial cell, a fungal cell, or the cell of a more complex organism, such as a human white blood cell.
During sample preparation, cells or viral particles may be treated chemically or enzymatically to dissolve or denature the membranes and proteinaceous coats, resulting in release of nucleic acids. This process of dissolution is commonly referred to as “lysis” and the resulting solution containing such lysed material is referred to as a “lysate” or “extract”. Unfortunately, such release exposes nucleic acids to degradation by endogenous nucleases present in the sample, which may exist in such abundance that destruction of the nucleic acids begins immediately upon release. Any nucleases remaining during subsequent purification processes can continue to degrade intact nucleic acids, resulting in the loss of the target molecule from the sample. Nucleases are abundant in most biological samples and are often extremely resistant to treatments known to inactivate other enzymes. In particular, ribonucleases (RNases) are active in most, if not all, biological samples, particularly blood, which is the most commonly collected tissue for diagnostic purposes. In addition to nucleases, many other proteins and contaminants are frequently present in biological samples and/or released during preparation of the lysate. For example, blood samples may include heme and heparin (an anti-coagulant used during blood drawing), which interfere with and/or inhibit, many downstream analytic procedures.
To overcome the problem of nucleases and other undesirable products present in biological lysates, it has been necessary to devise complex protocols with steps to both inactivate enzymes and to further purify nucleic acids. For example, anionic detergents and chaotropic agents, such as guanidinium thiocyanate, have been used to simultaneously inactivate or to inhibit nuclease activities while releasing nucleic acids from within cells and subcellular structures. To further purify nucleic acids from lysates, it is common in the art to precipitate the nucleic acids out of the solution, using a low molecular weight alcohol. Because other macromolecules also precipitate under these conditions, producing a sticky, intractable mass that entraps the nucleic acids, it has frequently been necessary to resort to extraction of the sample with hazardous organic solvent mixtures containing phenol, and/or chloroform prior to ethanol precipitation.
A further challenge in preparing samples for RNA analysis is protecting the integrity of these labile molecules. Unlike DNA, RNA is extremely susceptible to hydrolysis. As a consequence, although lysates containing DNA targets can be prepared from biological samples under harsh conditions, preparation of samples for RNA analysis has typically required the use of stabilizing agents, nuclease inhibitors and refrigeration and/or freezing. Variations of two methods have historically been used to prepare RNA from biological samples: chemical extraction and immobilization on glass, often referred to as “solid-phase extraction”. As described above, chemical extraction methods usually use highly concentrated chaotropic salts in conjunction with acidic phenol or phenol-chloroform solutions to inactivate RNases and purify RNA from other biomolecules. These methods provide very pure preparations of RNA; however, the RNA must typically be desalted and concentrated with an alcohol precipitation step. The solid-phase extraction method, described in U.S. Pat. No. 5,234,809 to Boom et al., relies on the lysing and nuclease-inactivating properties of the chaotropic agent guanidinium thiocyanate together with the nucleic acid-binding properties of solid-state silica particles or diatoms in the presence of this agent. After silica-bound RNA is washed with a high-salt buffer containing ethanol, the RNA is eluted in a low-ionic-strength buffer.
It will be readily appreciated that sample preparation methods requiring aqueous extraction with organic solvents or chaotropic agents are tedious, hazardous, labor-intensive, and slow. Moreover, if great care is not taken in performing the procedures, residual contamination with nucleases can occur, and the sample nucleic acids will be degraded or lost. Diagnostic tests performed with such samples can give false negative results due to such degradation. False negative results can also be obtained due to chemical interference, for example from residual anionic detergents, chaotropic salts, or ethanol remaining in the sample and inhibiting target amplification procedures. If anionic detergents and proteases have been used, residual proteolytic activity can also degrade the enzymes used in target amplification and/or hybridization detection reactions and produce false negative results. Sample preparation methods based on the “Boom lysis” protocol disclosed in the '809 patent are commonly viewed as adequately addressing these problems. However, the present inventors have unexpectedly found that such extraction methods, utilizing chaotropic salts combined with solid-phase extraction, are not reliably effective in the preparation of blood or plasma samples for PCR-based detection of the HBV genome. Thus, none of the above-cited protocols is suitable for the preparation of a common sample for detection of both DNA and RNA targets from complex biological starting materials, e.g., whole blood and blood serum. This is particularly true for infectious disease diagnosis in clinical laboratory settings, where time demands are very high, and in low-resource areas where cost-effectiveness, reduction of toxic waste streams and simplicity are also of prime importance.
While progress has been made in the field, there continues to be a need in the art for extraction of nucleic acids from various samples and methods for preparation of nucleic acid containing solutions. The present invention fulfills these needs and provides further related advantages.